Thrombomodulin has been known as a substance that acts to specifically bind to thrombin so as to inhibit the blood coagulation activity thereof, and at the same time, acts to significantly promote the ability of the thrombin to activate protein C. Thrombomodulin has also been known to have strong blood coagulation-inhibiting action. It has also been known that thrombomodulin extends the coagulation time by thrombin, or that it suppresses platelet aggregation due to thrombin. Protein C is a vitamin K-dependent protein that plays an important role in a blood coagulation fibrinolytic system. Protein C is activated by the action of thrombin, so that it becomes activated protein C. It has been known that the activated protein C inactivates an activated blood coagulation factor V and an activated blood coagulation factor VIII in vivo, and that it is involved in generation of a plasminogen activator having thrombolytic action (Non-Patent Document 1). Accordingly, it has been considered that thrombomodulin promotes the activation of protein C by thrombin, and thus that this is useful as an anticoagulant or a thrombolytic agent. Also, it has been reported in an animal experiment that thrombomodulin is effective for therapy or prevention of diseases associated with acceleration of coagulation (Non-Patent Document 2).
Conventionally, thrombomodulin has been discovered and obtained as a glycoprotein that is expressed on the vascular endothelial cells of various animal species, including humans as typical examples, and thereafter has been successfully cloned. That is to say, a human thrombomodulin precursor gene containing a signal peptide has been cloned from a human lung cDNA library by genetic engineering, and all the gene sequences of thrombomodulin have been analyzed. As a result, an amino acid sequence consisting of 575 residues containing a signal peptide (in general, 18 amino acid residues are exemplified) has been clarified (Patent Document 1). It has been known that a mature thrombomodulin, from which the signal peptide has been cleaved, is composed of 5 regions, namely, an N-terminal region (amino acids 1-226: this is the position determined when the signal peptide is assumed to consist of 18 amino acid residues, and the same holds true for other regions), a region having six EGF-like structures (amino acids 227-462), an O-linked glycosylation region (amino acids 463-498), a transmembrane region (amino acids 499-521), and an intracytoplasmic region (amino acids 522-557), from the N-terminal side of the mature peptide. It has also been known that, among the six EGF-like structures, the 4th, 5th, and 6th EGF-like structure portions from the N-terminal side (that is, minimal units of activity) mainly have the same activity as that of the entire-length thrombomodulin (Non-Patent Document 3).
Unless a surfactant is present, the entire-length thrombomodulin is hardly dissolved. Thus, addition of a surfactant is necessary for producing a thrombomodulin preparation. In contrast, there is also a soluble thrombomodulin that can be fully dissolved even in the absence of a surfactant. The soluble thrombomodulin may be prepared by removing at least a part of the transmembrane region or the entire transmembrane region. For example, it has been confirmed that a soluble thrombomodulin consisting of only 3 regions, namely, an N-terminal region, a region having six EGF-like structures, and an O-linked glycosylation region (that is, a soluble thrombomodulin having an amino acid sequence consisting of amino acids 19-516 of SEQ ID NO: 1), can be obtained by applying recombination techniques, and that this recombinant soluble thrombomodulin has the same activity as that of a native thrombomodulin (Patent Document 1). In addition, there are some other reports regarding soluble thrombomodulins (Patent Documents 2 to 9). Also, a human urine-derived soluble thrombomodulin and the like have been exemplified as native thrombomodulins (Patent Documents 10 and 11).
As recognized in many cases, as a result of spontaneous mutations or mutations occurring when thrombomodulins are obtained, polymorphic mutations have been found even in human genes. At present, thrombomodulin genes in which the amino acid at position 473 of a human thrombomodulin precursor having the aforementioned amino acid sequence consisting of 575 amino acid residues is converted to Val or Ala have been identified. In a nucleotide sequence encoding this amino acid, the nucleotide at position 1418 is converted to T or C (Non-Patent Document 4). However, the two thrombomodulins are completely identical in terms of their activity and physical properties. Thus, it can be considered that they are substantially identical.
It has been reported that thrombomodulin has effects on the therapy of DIC (Non-Patent Document 5). In addition to the aforementioned intended uses, it is anticipated that thrombomodulin will be used in the therapy and prevention of various diseases such as acute coronary syndrome (ACS), thrombosis, peripheral vascular obstruction, arteriosclerosis obliterans, vasculitis, functional disorder occurring after heart surgery, complication caused by organ transplantation, angina pectoris, transient ischemic attack, toxemia of pregnancy, diabetes, liver VOD (liver veno-occlusive disease; e.g. fulminant hepatitis, veno occlusive disease of liver occurring after bone marrow transplantation), deep venous thrombosis (DVT), and adult respiratory distress syndrome (ARDS).
As denatured products of thrombomodulin, an aggregate generated in a freeze-drying process and an aggregate generated during long-term preservation in a freeze-dried state have been known (Patent Documents 12 to 16).
Known methods for producing soluble thrombomodulin at an industrial level for use in pharmaceutical products include: a method using affinity chromatography in which an antibody reacting with thrombomodulin is supported in a purification step; a method for producing high-purity soluble thrombomodulin substantially containing neither serum-derived products nor antibody-derived products, which is characterized in that the soluble thrombomodulin is obtained as a pass-through fraction in a step of allowing the soluble thrombomodulin obtained by affinity chromatography to come into contact with a cation exchanger under conditions consisting of a specific conductivity of 25 to 34 ms/cm and pH 3 to 4 (Patent Document 17); and a method for purifying thrombomodulin, which is characterized in that it comprises preliminarily purifying a human urine thrombomodulin-containing sample by thrombin-bound affinity chromatography and then purifying the sample by adsorption chromatography using hydroxyapatite as an adsorbent (Patent Document 18).    Patent Document 1: JP Patent Publication (Kokai) No. 64-6219 A (1989)    Patent Document 2: JP Patent Publication (Kokai) No. 2-255699 A (1990)    Patent Document 3: JP Patent Publication (Kokai) No. 3-133380 A (1991)    Patent Document 4: JP Patent Publication (Kokai) No. 3-259084 A (1991)    Patent Document 5: JP Patent Publication (Kokai) No. 4-210700 A (1992)    Patent Document 6: JP Patent Publication (Kokai) No. 5-213998 A (1993)    Patent Document 7: WO92/00325    Patent Document 8: WO92/03149    Patent Document 9: WO93/15755    Patent Document 10: JP Patent Publication (Kokai) No. 3-86900 A (1991)    Patent Document 11: JP Patent Publication (Kokai) No. 3-218399 A (1991)    Patent Document 12: JP Patent Publication (Kokai) No. 6-321805 A (1994)    Patent Document 13: JP Patent No. 3007785    Patent Document 14: JP Patent Publication (Kokai) No. 11-171790 A (1999)    Patent Document 15: WO95/16460    Patent Document 16: JP Patent No. 3822383    Patent Document 17: JP Patent Publication (Kokai) No. 11-341990 A (1999)    Patent Document 18: JP Patent No. 3745805    Non-Patent Document 1: Koji Suzuki, Igaku no Ayumi (Progression of Medicines), Vol. 125, p. 901 (1983)    Non-Patent Document 2: K. Gomi et al., Blood 75, 1396-1399 (1990)    Non-Patent Document 3: M. Zushi et al., J. Biol. Chem., 264, 10351-10353 (1989)    Non-Patent Document 4: D. Z. Wen et al., Biochemistry, 26, 4350-4357 (1987)    Non-Patent Document 5: S. M. Bates et al., Br. J. of Pharmacol., 144, 1017-1028 (2005)